Nano- and/or micro-structured printed electrodes

ABSTRACT

The present invention relates to new electrochemical probes for the measurement of an analyte selected from the group consisting of: free chlorine, chlorine dioxide, total chlorine and peracetic acid, wherein said probe includes at least a nano- or micro-structured printed electrode with a nano- or micromaterial selected from the group consisting of: nano- or microparticles of carbon black and/or nano- or microparticles of a metal selected from the group consisting of gold, silver, platinum, copper and combinations or alloys thereof.

FIELD OF THE INVENTION

The present invention relates to new electrochemical sensors and probescomprising one or more of said sensors, useful for the measure in afluid of an analyte selected from the group consisting of: freechlorine, chlorine dioxide, total chlorine and peracetic acid;characterized in that said sensor includes at least a printed electrodenano- or micro-structured with a nano- or micromaterial selected fromthe group consisting of: nano- or microparticles of carbon black and/ornano- or microparticles of a metal selected from the group consisting ofgold, silver, platinum, copper and combinations or alloys thereof;useful for monitoring water pollution and/or compounds useful fordisinfecting water for domestic or industrial use, or water for swimmingpools.

The present invention also relates a kit of an integrated system for themanagement of the sensors of the invention, and the use of suchintegrated system or kit for monitoring water pollution and/or compoundsuseful for the disinfection of domestic, industrial and swimming poolwater.

BACKGROUND OF THE INVENTION

The drinking water biological pollutants represent still today thecompounds responsible for acute infectious diseases.

Therefore it is necessary to carry out a disinfection treatment ofwaters destined to human consumption.

Chlorine dioxide was initially used as whitener in paper industry; sincethe 1950s it has also been employed as disinfectant and algaecide. Thedisinfectant properties of chlorine dioxide remain unaltered over a widepH range and this product does not significantly alter the organolepticcharacteristics of the water in which it is added.

“Free chlorine” is defined as the sum of the concentrations of thehypochlorite ion and of the hypochlorous acid, which both are producedby the reaction of hydrolysis of sodium hypochlorite, gaseous chlorine,calcium hypochlorite and isocyanuric acid derivatives (sodiumdichloroisocyanurate and trichloroisocyanuric acid).

“Total chlorine” is defined as the sum of inorganic free chlorine andorganic/inorganic combined chlorine. When nitrates of organic originand/or ammonia compounds are present in water, inorganic chlorine reactsforming chloramines and its presence constitutes the combined chlorine.Combined chlorine can be classified in combined inorganic chlorine,derived from the reaction with ammonia, and in combined organicchlorine, obtained from the reaction between chlorine and nitrogencompounds, such as amino acids.

Peracetic acid is a liquid organic compound with a characteristicpungent odor, mainly employed as disinfectant in food, cosmetics andpharmaceuticals industries.

Its use for the treatments of large plant surfaces has certainlygenerated a considerable interest, thanks to its capability to easilysolve some management problems, such as the bacteria abatement in shorttimes at room temperature, and the absence of by-products harmful tohuman health.

However, peracetic acid also presents negative aspects concerning itsinstability, though it definitely remains one of the most useddisinfection products on industrial scale. Actually, peracetic acidsolution is available on market with different concentrations; those at5% and 15% w/w are the most used.

To evaluate whether the concentration of disinfectants can be sufficientto guarantee an adequate disinfection, and at the same time notexcessively high to produce reaction by-products harmful to humans intreated water, the concentration of such disinfectants must bemonitored.

To date many instruments for the determination of free chlorine, whichuse the colorimetric or electrochemical detection, are available on themarket.

In particular, the electrochemical probes present on the market areconstituted from classical gold electrodes coupled to Ag/AgX referenceelectrodes, X=halide (http://www.etatronds.it;https://www.prominent.it/it). These sensors present severaldisadvantages. First, they are very expensive because bulk gold is usedto realize the working electrode. Furthermore, there is the need toclean the surface of the working electrode after some measures.

In literature, different studies have been reported with the purpose ofdeveloping electrochemical sensors for the determination of freechlorine. For example, in Analytica Chimica Acta (2005), 537: 293-298,platinum, gold and glassy carbon electrodes performances were compared,achieving a detection limit (LOD) of 1 ppm by using a platinumelectrode. These kinds of electrodes have the disadvantage of undergoinga passivation process of the electrode surface in the presence of highconcentrations of free chlorine. Furthermore, the sensor configurationdoes not allow an easy miniaturization (Analytica Chimica Acta (2005),537: 293-298). In order to develop miniaturized electrodes, in Talanta[(2016), 146: 410-416], screen-printed carbon sensors, requiring theelectrodeposition of Prussian Blue are described, thus making difficultan easy mass production of the sensors themselves.

With reference to free chlorine, the Italian Institute of Healthestablishes that spectrophotometric detection is the reference methodfor free chlorine determination. Chlorine, in fact, oxidizing a solutionof N,N-diethyl-p-phenylenediamine (DPD), leads to the production of theWurster compound, whose absorbance at 510 nm is related to theconcentration of free chlorine in the sample.

However, high concentrations of the analyte produce a colorless andunstable imine formation, which causes a linearity loss of thecalibration curve above 1 ppm of oxidant. Furthermore, this methodallows the determination of the analyte in a range of 0.05-4 ppm(Current Technology of Chlorine Analysis for Water and Wastewater (2002)17: 2-11; Ottaviani M, Bonadonna L. (Ed.). Metodi analitici diriferimento per le acque destinate al consumo umano ai sensi del DL. Vo31/2001. Metodi chimici. Roma: Istituto Superiore di Sanità; 2007.(Rapporti ISTISAN 07/31).

Electrochemistry Communications 47 (2014) 63-66 describes an electrodefor measuring hydrogen peroxide, in which the printed electrode isfunctionalised with carbon black and nanoparticles of Prussian blue.

U.S. Pat. No. 6,627,058 refers to an electrode for measuring glucose, inwhich the printed electrode is functionalised with carbon black andnanoparticles of Prussian blue.

Microchim Acta (2016); Vol 183; #10; 2799-2806 reports an electrode formeasuring hydrogen peroxide in which on the printed electrode arepresent silver nanoparticles, and said electrode is functionalized byusing reduced graphene and cerium IV.

US 2014/083864A1 mentions a sensor useful for measuring, among theothers, chlorine or peracetic acid, in which the printed electrode iscovered with an ink containing conductive metal particles. In thispatent application it is never mentioned nor suggested the use of nano-or micro-structured metallic particles, which would have increased thecharacteristics of the electrode.

DE 4319002 describes a sensor for the measurement of peracetic acid, inwhich on the printed electrode platinum microparticles may be present.

In recent years, research in the field of sensors has been focused onthe production of screen-printed electrodes modified with nano- and/ormicromaterials (nano- and microsensors). Among the technologies used forthe production of screen-printed, screen printing technique is the mostsuitable technique for mass production of screen-printed electrodes withreduced costs.

In many scientific publications, sensors based on the use ofscreen-printed electrodes using screen printing technique, also modifiedwith nanomaterials for the measurement of different analytes indifferent matrices are described (Microchimica Acta (2015) 182: 643-651;Electroanalysis (2014) 26: 931-939; Electroanalysis (2015) 27:2230-2238; Microchemistry Acta (2016) 183: 2063-2083).

It is known in the state of the art that the production of stabledispersions of carbon nanotubes, for “sensors” preparation, requires theuse of strong acids such as nitric acid and strongly oxidizingsubstances such as the permanganate, which generate products thatrequire proper disposal.

To date in the field of water quality control there is still a strongperceived need to have available a system for monitoring free chlorine,total chlorine, chlorine dioxide, and peracetic acid that:

-   -   is integrated into a miniaturized system;    -   is low-cost;    -   is suitable for in situ application;    -   is easy to use even by unskilled person;    -   during the production processes does not require the use of        strong acids and/or agents strongly oxidizing that generate        highly polluting products that must be properly disposed.

DESCRIPTION OF THE INVENTION

It is therefore an object of the present invention a printed electrode,nano- and/or micro-structured, for the measure, in fluids, of an analyteselected from the group consisting of: chlorine dioxide, free chlorine,total chlorine and peracetic acid, characterized in that it is preparedby using nano- or microparticles of carbon black and/or nano- ormicroparticles of a metal selected from the group consisting of gold,silver, platinum, copper and combinations or alloys thereof.

It is a further object of the invention an electrochemical sensorcomprising at least one printed electrode as defined before. Inparticular, such sensor comprises

-   -   at least an “printed electrodes group”, in which said “printed        electrodes group” comprises at least a working electrode; at        least a reference electrode; and at least an auxiliary        electrode;

and is characterized in that:

-   -   the working electrode is functionalised/activated/prepared using        nano- or microparticles of carbon black; and/or nano- or        microparticles of a metal selected from the group consisting of        gold, silver, platinum, copper and combinations or alloys        thereof, preferably gold having an average diameter of 1 μm, in        which the amount of the nano- or microparticles of carbon black,        and/or metallic particles deposed on the working electrode is        from 0.1 to 50 μl, preferably from 1 to 20 μl, more preferably        is from 2 to 10 μl, and in which the deposition (on the working        electrode) is made by consecutive applications of 2 μl each.

According to the invention, the reference electrode and the counterelectrode may be located on the other side of the printed electrode,i.e. exposed to the reservoir (in other words, different electrode indifferent side of the printed electrode).

It is a further object of the present invention an electrochemicalsensor nano- and/or micro-structured;

comprising at least an “printed electrodes group” containing at least aworking electrode; at least a reference electrode; and at least anauxiliary electrode; preferably said printed electrodes group is furthercharacterized in that it comprises at least one hole (21) that allow thegel contained in the reservoir (12) to pass through and to act ascontacting electrolyte (see FIG. 1d ).

Preferably, the working electrode is activated/prepared with metalmicroparticles selected from the group consisting of gold, silver,platinum, copper and combinations or alloys thereof, having an averagediameter of from 20 to 0.05 μm; preferably from 10 to 0.3 μm; morepreferably (about) 1 μm;

and

-   -   the auxiliary electrode is prepared, during the process of        printing, using an ink containing a carbon based material,        preferably graphite;

or,

-   -   the working electrode (belonging to the “printed electrodes        group”) at the end of the printing process as described, for        example, in Analytica Chimica Acta 707 (2011) 171-177; is        functionalised by “drop-casting” as described, for example, in        Electroanalysis 24 (2012) 743-751;

by using:

-   -   nanomaterials selected from the group consisting of carbon black        or metallic nanomaterials selected from the group consisting of        gold, silver, platinum, copper and combinations or alloys        thereof; preferred are nanoparticles of gold, obtained, for        example, as described in Sensors and Actuators B (2015) 212:        536-543;

in which the amount of nano- or microparticles of carbon black, ormetallic particles deposed on the working electrode is from 0.1 to 50μl; preferably from 1 to 20 μl; more preferably from 2 to 14 μl;

and in which the deposition is carried out in consecutive applicationsof 2 μl each.

The printed electrodes obtained by drop-casting, with the process abovedescribed are further characterized in that:

-   -   the measure of free chlorine is made applying to the electrodes        a potential from −0.2 to +0.4 V; preferably from −0.2 to +0.1 V;        most preferably −0.1 V vs Ag/AgCl;    -   the measure of chlorine dioxide is made applying to the        electrodes a potential from +0.02 to +1 V; preferably from +0.01        to +0.5 V; most preferably +0.1 V vs Ag/AgCl;    -   the measure of total chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V, most        preferably from +0.3 to +0.6 V vs. Ag/AgX;    -   the measure of peracetic acid is made applying to the electrodes        a potential from −0.3 to 0 V; preferably from −0.1 to 0 V; most        preferably −0.1 V vs Ag/AgCl;

while the probes or sensors obtained by using the ink containing metalmicroparticles, according to the process above described, arecharacterized in that:

-   -   the measure of free chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V; most        preferably +0.5 V vs Ag/AgX;    -   the measure of chlorine dioxide is made applying to the        electrodes a potential from 0 to +1 V; preferably from +0.2 to        +0.8 V; most preferably from +0.3 to +0.5 V vs Ag/AgX;    -   the measure of total chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V, most        preferably from +0.3 to +0.6 V vs. Ag/AgX;    -   the measure of peracetic acid is made applying to the electrodes        a potential from −0.5 to 0 V; preferably from −0.3 to 0 V; most        preferably −0.2 V vs Ag/AgCl.

It is a further object of the present invention a probe comprising oneor more electrochemical sensor above described.

It is a further object of the present invention is a method forpreparing an electrochemical sensor nano- and/or micro-structured;comprising:

at least an “printed electrodes group” containing at least a workingelectrode; at least a reference electrode; and at least an auxiliaryelectrode; in which, preferably. said printed electrodes group ischaracterized in that it comprises at least one hole (21) that allow thegel contained in the reservoir (12) to pass through and to act ascontacting electrolyte (see FIG. 1d ).

Preferably, the working electrode and the reference electrode areprepared, during the process of printing, using an ink containing ametal microparticles selected from the group consisting of gold, silver,platinum, copper and combinations or alloys thereof, having an averagediameter of from 20 to 0.05 μm;

preferably from 10 to 0.3 μm; more preferably (about) 1 μm.

-   -   and        -   the auxiliary electrode is prepared, during the process of            printing, using an ink containing a carbon based material;            preferred is graphite;    -   or (alternatively):        -   the working electrode (belonging to the “printed electrodes            group”) at the end of the printing process is functionalised            by “drop-casting” by using:        -   nanomaterials selected from the group consisting of carbon            black or metallic nanomaterials selected from the group            consisting of gold, silver, platinum, copper and            combinations or alloys thereof; preferred are nanoparticles            of gold, obtained, for example, as described in Sensors and            Actuators B (2015) 212: 536-543;

in which the amount of nano- or microparticles of carbon black, ormetallic particles deposed on the working electrode is from 0.1 to 50μl; preferably from 1 to 20 μl; more preferably from 2 to 14 μl;

and in which the deposition is carried out in consecutive applicationsof 2 μl each.

The printed electrodes obtained by drop-casting, with the process abovedescribed are further characterized in that:

-   -   the measure of free chlorine is made applying to the electrodes        a potential from −0.2 to +0.4 V; preferably from −0.2 to +0.1 V;        most preferably −0.1 V vs Ag/AgCl;    -   the measure of chlorine dioxide is made applying to the        electrodes a potential from +0.02 to +1 V; preferably from +0.01        to +0.5 V; most preferably +0.1 V vs Ag/AgCl;    -   the measure of total chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V, most        preferably from +0.3 to +0.6 V vs. Ag/AgX;    -   the measure of peracetic acid is made applying to the electrodes        a potential from −0.3 to 0 V; preferably from −0.1 to 0 V; most        preferably −0.1 V vs Ag/AgCl;

while the probes or sensors obtained by using the ink containing metalmicroparticles, according to the process above described, arecharacterized in that:

-   -   the measure of free chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V; most        preferably +0.5 V vs Ag/AgX;    -   the measure of chlorine dioxide is made applying to the        electrodes a potential from 0 to +1 V; preferably from +0.2 to        +0.8 V; most preferably from +0.3 to +0.5 V vs Ag/AgX;    -   the measure of total chlorine is made applying to the electrodes        a potential from 0 to +1 V; preferably from +0.2 to +0.8 V, most        preferably from +0.3 to +0.6 V vs. Ag/AgX;    -   the measure of peracetic acid is made applying to the electrodes        a potential from −0.5 to 0 V; preferably from −0.3 to 0 V; most        preferably −0.2 V vs Ag/AgCl.

Another object of the invention is a kit comprising at least anelectrochemical probe as described before and further comprising:

-   -   at least a probe holder;    -   at least a capsule;    -   at least an electrolytic solution or gel;    -   at least an hydrophobic and/or hydrophilic membrane; and    -   at least an electronic control, implementation and/or data        transfer.

Such a kit is useful to make for continuous measurements in a way that askilled in the art knows very well.

According to the present invention, the sensor comprises a printedelectrode, as defined before, and preferably it consists of a workingelectrode, a reference electrode, a counter electrode and an electronicdevice that has the task to configure the electrodes group and toacquire and decode the current signal coming from the electrode group,in which the output signal can be a signal in voltage and/or currentand/or digital and/or LAN and/or radio frequency connection.

The sensor according to the invention is suitable for being used forsingle analytical determinations (see FIG. 1a ) or to be inserted inline for continuous monitoring of the analytes (see FIGS. 1b and 1c ).

DESCRIPTION OF THE FIGURES

In FIG. 1a it is shown the probe according to the present invention,useful for a single detection of tan analyte, comprising:

-   -   the printed electrodes group (1);    -   the measurement display (2);    -   the central body of the device (3);    -   the container of the liquid to be analyzed (4).

In FIG. 1b the probe according to the invention is presented, for acontinuous detection of the analyte under examination.

FIG. 1c shows a sectional detail of the probe according to theinvention, for a continuous detection of the analyte under examination,comprising:

-   -   a capsule supporting structure (9);    -   a printed sensor (working electrode or reference electrode or        auxiliary electrode) (10);    -   a hydrophobic or hydrophilic membrane (11);    -   an electrolyte gel solution (12);    -   a sensor body (13), containing the electronic management or        system of the sensor;    -   contacts (14) for connection to the capsule;    -   contacts (15) for connection to the sensor body (13);    -   a connection cable of the screen-printed electrode (16) (in        total the connection cables are three, one for each electrode);    -   a separating septum (20).

FIG. 1d shows the printed sensor (working electrode/referenceelectrode/auxiliary electrode) (10), in which some holes (21) arepresent, that allow the gel contained in the reservoir (12) to passthrough and to act as contacting electrolyte.

FIG. 1e shows an example of a probe holder useful to contain at leastone probe object of the present invention, in which:

(17) represents a flow meter (for the control of flow parameters);

(18) represents the pH probe or electrode known in the art; and

(19) represents the probe or electrode according to the presentinvention.

It is evident to the expert of the art that a probe holder may consistof a single module (19), if a flow parameter control is carried outupstream; otherwise it may consist of module (17) and (19); or of module(17), (19) and at least a further module in which to insert one or moreprobes for further measurement of analyte.

FIGS. 2a and 2b show the printed electrodes group (1), both disassembled(FIG. 2a ) and assembled (FIG. 2b ) comprising:

-   -   a flexible substrate or support (5), on which the electrode is        printed;    -   three metal conductive tracks (6), one of which at its end        constitutes the reference electrode (6 a);    -   three graphite tracks (7) (and/or other suitable material, known        in the art), one of which at its end is nano- or        micro-structured according to the invention and constitutes the        working electrode (7 a) and another one of which at its end        constitutes the auxiliary electrode (7 b); and    -   an insulating layer (8).

Figures obtained by electron microscopy of the printed electrodes,before (FIGS. 3a and 3b ) and after (FIGS. 3c and 3d ) the modificationby drop-casting with the dispersion of Carbon Black (CB), are shown. InFIGS. 3c and 3d the deposition of CB nanoparticles that completely coverthe working electrode surface is clearly evidenced.

FIG. 4 shows the control and/or actuation unit (known in art) which hasthe function of collecting data detected by the various peripheralprobes and to activate the dosing pumps for modifying this data, if any.The control of these parameters (free chlorine, chlorine dioxide, totalchlorine, peracetic acid and/or pH) can be made manually orautomatically, locally or using a remote control of the parameters.

The presence of a display, optionally touchscreen, locally or in remote,improves the management of the “system”; for further information aboutcontrol and/or implementation unit, the following link may be useful:

http://www.etatronds.it/dettaglio_prodotto.php?id=187&super=18&cat=ESELECT

FIGS. 5a, 5b, 5c and 5d report:

-   -   the trend in amperometric responses of electrodes modified with        2, 4, 6, 8 and 10 μl of CB (2 μl for n times) at a fixed        potential of 0.25 V vs Ag/AgCl for sodium hypochlorite (free        chlorine) concentrations ranging from 0.1 to 10 60 ppm (FIG. 5a        ). The sensitivity (black bars) and the RSD % (gray bars) are        reported in FIG. 5a . Increasing the amount of CB nanoparticles        deposited on the working electrode, an increase in the        amperometric response has been observed. A volume of CB of 10 μl        has been chosen, as this value allowed obtaining the best result        in terms of repeatability, signal stability and sensitivity.    -   the trend in the amperometric responses by varying the range of        potential from −0.2 V to 0.4 V vs Ag/AgCl (FIG. 5b ). The best        result in terms of sensitivity was obtained by applying a        potential of −0.1 V vs Ag/AgCl with an RSD % inter-electrode        equal to 6%;    -   the trend in the amperometric responses by varying pH in a range        between 2 and 12 (FIG. 5c ). pH-value 5 permitted to obtain a        sensitive and accurate measurement;    -   the trend in the amperometric responses following ionic strength        changes (FIG. 5d ). A Britton-Robinson buffer 0.02 M+KCl 0.02 M        resulted the best solution to be used.

FIGS. 6a and 6b show the data obtained (amperometric curve with relativeequation and calibration curve-insert) related to the inter-electrode(FIG. 6a ) and intra-electrode (FIG. 6b ) repeatability.

In particular, the measurements carried out using same electrode ordifferent electrodes gave the similar response, demonstrating therepeatability of the system.

The calibration curve was obtained by reporting the mean value (n=3) ofcurrent recorded in function of the hypochlorite concentration inBritton Robinson buffer solution 0.02 M+KCl 0.02 M pH=5, applying duringamperometric tests a potential of −0.1 V.

From the results obtained, excellent inter-electrode and intra-electroderepeatability was observed; the same sensor was able to detect achlorine concentration range between 0.05 and 200 ppm.

FIG. 7 shows the results obtained in the interference study byevaluating possible interfering ionic species (NO³⁻, SO₄ ²⁻, CO₃ ²⁻,HCO₃ ⁻and Cl⁻ions). These ions may be present in swimming pool waterduring maintenance treatments. The results obtained show that thepresence of the ions did not modify the sensor response compared to theanalyte and, above all, the sensor(s) did not show an electrochemistryresponse against them.

In FIGS. 8a and 8b results obtained for the chlorine dioxide sensor arereported; in details, the voltammetric study in B.R. 0.02 M+KCl 0.02 M,pH=2 in the absence (dashed line) and in the presence (continuous line)of analyte with unmodified sensor (black line) and modified with CB(gray line) (FIG. 8a ); in this case the standard solution of chlorinedioxide was prepared using the reagent h.

FIG. 8b reports the results obtained from the amperometric study carriedout by applying a potential of 0 V vs AgCl and using a sensor modifiedwith various amounts of CB (FIG. 8b ).

In FIGS. 9a and 9b , data obtained from the study of dioxide chlorineconcentration as a function of pH in buffer B.R 0.02 M+KCl 0.02 M byspectrophotometric analysis (black bars, instant concentration, graybars, after 24 h) (FIG. 9a ) are showed.

In FIG. 9b the cyclic voltammetry study to confirm the dataspectrophotometrically obtained in B.R. 0.02 M+KCl 0.02 M is shown, inthe absence (dashed line) and in the presence of analyte (solid line) atpH 4 (gray line) and pH=2 (black line).

In FIG. 10, the inter-electrode amperometric study for the measurementof chlorine dioxide obtained with three different electrodes (lightgray, dark gray and black amperogramms showed the response for eachdifferent electrode) is reported, by applying a potential of 0.1 V vsAg/AgCl in Britton-Robinson buffer 0.02 M+KCl 0.02 M pH=2. Insert:calibration line obtained as an average of the currents obtained fromthree different electrodes as a function of the concentration of theanalyte (0.1-10 ppm); using “the reagent h” for preparing the standardsolution of chlorine dioxide.

In FIGS. 11a, 11b, 11c and 11d results obtained from amperometricstudies by the sensor for the measurement of peracetic acid, conductedfor the choice of operating parameters, are reported:

-   -   optimization of the amount of gold nanoparticles deposited on        the working surface in 0.1 M acetate buffer, pH=5.4 and E=−0.1        V,    -   optimization of the potential applied with 6 μL gold        nanoparticles in 0.1 M acetate buffer and pH=5.4,    -   pH optimization with 6 μL gold nanoparticles in buffer acetate        0.1 M and E=−0.1 V,    -   optimization of the ionic strength of the working solution with        6 μl of gold nanoparticles in acetate buffer pH=5.4 and E=−0.1.

FIG. 12 shows a block diagram of the system in which the sensor wasoptimized and integrated with a potentiostat, developed to allow anautomatic measurement of free chlorine. Inside the probe, a block thatprovided for conditioning, processing and transmitting data coming fromthe electrodes using electrical diagrams and software well known in theart was present.

FIG. 13 shows the communication system between the sensor and theoperational amplifier. In fact, the signal conditioning system providedthe use of operational amplifiers to keep the voltage constant and tomeasure the current.

The operational amplifier was able to decouple the control system of themicrocontroller from the measurement system.

FIG. 14 shows the communication system between microcontroller andoperational amplifier. The processing system provides a continuous dataexchange between the microcontroller and the operational amplifiersthrough ADC and 12 bit DAC. The microcontroller, using the DAC,continuously provides the working voltage to be applied to theelectrodes. The operational amplifier is used to transform the currentcoming from the electrodes. This current is changed into voltage andmeasured through the ADC present on the micro.

A signal is generated at the output of the electronic system, which inturn is sent to the control and/or to the implementation control unit.This signal is proportional to the measured analyte concentration.

The potentiostat circuit and the microcontroller that acquires thesignal in current coming from the electrode assembly (well known in theart and easily replicable by a sector expert), constitute a system thatallows the measurement of particular analyte concentrations; the devicethat manages the system logic is a microcontroller well known in theart.

In FIG. 15, the operational algorithm of the program, easily written bya technician skilled in the art, is reported. The main program (see alsoFIG. 12) provides, after the Pin assignment, the variables declarationused within it, including the working voltage variable to which theelectrodes are subjected. After setting the working voltage variable,the program will start measuring the current between the electrodesproviding an input voltage to the microcontroller. The program alsoallows choosing the type of output signal.

The examples below illustrate the invention without limiting it.

Materials and Methods Instruments

Magnetic stirrer, Hanna instruments

Digital pH-meter 334-B, Amel Instrument

Analytical balance, Sartorius

High performance multipurpose precision screen printer DEK 245, DEK,Weymouth, UK

Sonicator Falc

Portable potentiostat/galvanostat PalmSens Instrument, Eco Chemie,Utrecht, Olanda con software PSTrace 4.4

Reagents

a) Graphite ink, Electrodag 423 SS

b) Silver ink, Electrodag 477 SS

c) Gold ink, Dupont BQ331

d) Insulating ink, Gwent Group D2070423P5

e) Insulating ink, Electrodag PF 455B

f) Polyester substrate, Autostat HT5

g) Gold nanoparticles, Stream Chemicals Ref. 79-0180.

h) Chlorine dioxide release mixture Aldrich chemical Company,[7758-19-2]

EXAMPLE 1 Preparation Procedure for a Functionalized Sensor Useful forthe Free Chlorine Measurement Using Drop-Casting Method

For the preparation of the functionalized sensor for free chlorinemeasurement, as “starting product”, a not functionalized sensor orelectrode was used; the functionalization was carried out by depositingon the surface of the working electrode 10 μl (5 depositions of 2 μleach) of a dispersion of CB (Carbon Black N 220 from Cabot RavennaItaly); the dispersion was prepared by placing 1 mg of CB in 1 ml of awater and dimethylformamide solution (1: 1); before use, this dispersionwas sonicated for an hour at 59 KHz; with obtaining a functionalizedelectrode for the detection of free chlorine.

The liquid to be analyzed (for the detection of free chlorine), beforereading, was placed in a working solution, consisting of aBritton-Robinson buffer+KCl at pH 5, with an ionic strength of 0.02 MBritton- Robinson and 0.02 M for the KCl. For the measure a potential of−0.1 V vs Ag/AgCl was applied.

The free chlorine electrode according to the invention was characterizedby the analytical point of view to determine the linear range,sensitivity and inter- and intra-electrode repeatability. Thecalibration curve was obtained by reporting the mean value (n=3) ofcurrent recorded as a function of free chlorine concentration in BrittonRobinson buffer solution 0.02 M+KCl 0.02 M pH=5, applying, during theamperometric tests, a potential of −0.1 V, obtaining a sensitivity of0.32±0.02 μA/ppm and RSD %=6%. The results obtained show an excellentinter- (FIG. 6a ) and intra-electrode repeatability (FIG. 6b ); thesensor according to the invention was able to detect a free chlorineconcentration range between 0.05 and 200 ppm.

A similar response was also observed using trichloroisocyanuric acid asstandard: 0.36±0.01 μA/ppm and RSD %=3%.

The limit of detection (LOD) of the sensor according to the invention,calculated as S/N=3, was equal to 0.01 ppm and the LOQ=0.03 ppm obtainedfrom S/N=10.

EXAMPLE 2 Preparation Procedure for a Functionalized Sensor Useful forthe Free Chlorine Measurement, Where the Working Electrode and theReference Electrode are Prepared, During the Process of Printing, Usingan Ink Containing a Metal (the Auxiliary Electrode is Prepared withMethods Known in the Art Using an Ink Containing Graphite

For the preparation of the functionalized sensor for free chlorinemeasurement, the non-functionalized sensor or electrode was used as“starting product”, for which an ink based on gold microparticles wasused for working electrode.

For the measurement, an electrolyte solution consisting of a buffersystem, preferably phosphate, borate, acetate, citrate, or mixturesthereof, was used, based on the field of application of the sensor, morepreferably a buffer which maintains the pH value in a range from 2 to 12is used, a supporting electrolyte preferably a halogenated salt inrelation to the type of reference electrode of the sensor and to theanalyte to be determined at a variable concentration more preferablybetween 1% and 15%; and if necessary.

For the measurement to the electrodes a potential of +0.5 V vs Ag/AgXwas applied.

Using this electrode, we moved to the analytical characterization inorder to determine the range/linearity interval and inter-electroderepeatability that were 0-20 ppm and 2%, respectively. Furthermore, thesensitivity turned out to be 400 nA/ppm.

EXAMPLE 3 Preparation Procedure for a Functionalized Sensor for theMeasurement of Chlorine Dioxide Using Drop-Casting Method

For the preparation of the functionalized sensor, as “starting product”,a non-functionalized the sensor was used; the functionalization wascarried out using 2 μl of carbon black nanoparticles (prepared asdescribed in Example 1) (FIG. 8).

For the measurement, a working solution consisting of Britton-Robinsonbuffer+KCl at pH 2 (FIG. 9) was used with an ionic strength of 0.02 Mboth for Britton-Robinson and for KCl (FIG. 9); applying to theelectrodes a potential of +0.1 V vs Ag/AgCl.

The chlorine dioxide standard solution was prepared using the reagent h,chlorine dioxide release mixture,

Using the operating parameters described above, a functionalizedelectrode was obtained.

The chlorine dioxide electrode, according to the invention, wascharacterized by the analytical point of view to determine the linearrange, sensitivity and inter- and intra-electrode repeatability. Thecalibration curve was obtained reporting the mean value (n=3) of currentrecorded as a function of the concentration of dioxide chlorine inBritton Robinson buffer solution 0.02 M+KCl 0.02 M, pH 2, applyingduring the amperometric tests a potential of +0.1 V with a sensitivityof 13.4±0.3 nA/ppm (FIG. 10).

From the obtained data, an excellent inter-(RSD %=2.2) intra (RSD %=2.7)electrode repeatability was observed; the sensor, according to theinvention, was able to detect a chlorine dioxide concentration rangebetween 0.1 and 10 ppm.

The limit of detection (LOD) of the sensor according to the invention,was equal to 0.03 ppm and the LOQ=0.1 ppm.

Finally, the sensor according to the invention also proved its validityin pool water. Because pool water is a complex matrix, it was necessaryto dilute the sample and the dilution factor chosen, as a compromisebetween sensitivity and low matrix effect, it was equal to 1:5 v/v inbuffer solution. The sensitivity obtained was 5.4±0.4 nA/ppm. Theaccuracy of the sensor was evaluated using the recovery method,obtaining a percentage recovery of 78±8%.

Alternatively, it is possible to synthesize the standard solution ofchlorine dioxide using sodium chlorite and hydrochloric acid, thefunctionalized electrode, useful for the determination of chlorinedioxide, showed an improved sensitivity equal to 278±65 nA/ppm.

EXAMPLE 4 Preparation Procedure for a Functionalized Sensor for theMeasurement of Chlorine Dioxide Where the Working Electrode and theReference Electrode are Prepared, During the Process of Printing, Usingan Ink Containing Metal

For the preparation of the functionalized sensor for the measurement ofchlorine dioxide, the non-functionalized sensor or electrode was used,as “starting product”, for whose working electrode an ink based on goldmicroparticles was used.

For the measurement, an electrolyte solution, consisting of a buffersystem, preferably phosphate, borate, acetate, citrate, or mixturesthereof, was used, based on the field of application of the sensor, morepreferably a buffer is used which maintains the pH value in a range from2 to 12, a supporting electrolyte preferably a halogenated salt inrelation to the type of reference electrode of the sensor and to theanalyte to be determined at a variable concentration more preferablybetween 1% and 15%; and if necessary.

For the measure a potential of +0.3 to +0.5 V vs Ag/AgX was applied.

The chlorine dioxide electrode according to the invention, wascharacterized by the analytical point of view to determine the linearrange and inter electrode repeatability, obtaining a linear range up to10 ppm with a good inter-electrode repeatability (RSD %=5%).

EXAMPLE 5 Preparation Procedure for a Functionalized Sensor forMeasurement of Total Chlorine Using Drop-Casting Method

For the preparation of the functionalized sensor for total chlorinemeasurement, as “starting product”, the non-functionalized sensor wasused; the functionalization was carried out using 6 μl of goldnanoparticles (reagent g).

For the measurement, an electrolyte solution consisting of a buffersystem, preferably phosphate, borate, acetate, citrate, or mixturesthereof, was used, based on the field of application of the sensor, morepreferably a buffer is used which maintains the pH value in a rangebetween 2 and 8), a supporting electrolyte preferably a halogenated saltin relation to the type of reference electrode of the sensor and to theanalyte to be determined at a variable concentration more preferablybetween 1% and 15%; and if necessary.

For the measure a potential of +0.3 to +0.6 V vs Ag/AgX was applied.

The total chlorine electrode according to the invention, wascharacterized by the analytical point of view to determine the linearrange, and inter-electrode repeatability, obtaining a linear range up to20 ppm with a good inter-electrode repeatability (RSD %=5%).

EXAMPLE 6 Preparation Procedure for a Functionalized Sensor forMeasurement of Total Chlorine Where the Working Electrode and theReference Electrode are Prepared, During the Process of Printing, Usingan Ink Containing Metal

The screen-printed electrode for the sensor useful for the measurementof total chlorine was functionalized during the printing process usingink based on gold microparticles with an average diameter of 1 μm(reagent c).

For the reading, an electrolytic solution or gel consisting of a buffersystem, preferably phosphate, borate, acetate, citrate and mixture ofthem was used, chosen in accordance with the scope of the sensor, morepreferably a buffer that can maintains the pH value in an inclusiverange between 2 and 12, a supporting electrolyte preferably ahalogenated salt in relation whit the type of reference electrode of thesensor and to the analyte to be determined at a variable concentration,more preferably between 1% and 15%; and if necessary, in accordance withthe type of membrane used, a gelling agent chosen from the family oforganic compounds of natural origin, miscible in water in percentageranging from 85% to 100%.

For the reading, a potential of 0.3 to 0.6 vs Ag/AgX was applied to theelectrodes.

The total chlorine electrode according to the invention, wascharacterized by the analytical point of view to determine the linearrange, sensitivity and inter- and intra-electrode repeatability,obtaining a linear range up to 20 ppm with a good inter-electroderepeatability (RSD %=6%).

EXAMPLE 7 Preparation Procedure of a Functionalized Sensor Useful forthe Measurement of Peracetic Acid Using Drop-Casting Method

For the preparation of the functionalized sensor for measuring the acidperacetic, as “starting product”, the not functionalized sensor wasused; the functionalization was carried out using 6 μl of a dispersionof gold nanoparticles (FIG. 11a ) with a diameter of 5 nm commerciallyavailable, purchased from Strem Chemicals, n. of catalog 79-0180(reagent g), and applying a potential of −0.1 V vs Ag/AgCl (FIG. 11b ).

For the measurement, a working solution consisting from an acetatebuffer 0.1 M at pH 5.4 was used (FIG. 11c ), and an ionic strength of0.1M (FIG. 11d ); applying to the electrodes a potential of −0.1 V vsAg/AgCl.

Using operating parameters described above, a functionalized electrodewas obtained.

The peracetic acid electrode according to the invention, wascharacterized by the analytical point of view to determine the linearrange, sensitivity and inter- and intra-electrode repeatability. Thecalibration curve was obtained by reporting the mean value (n=3) ofcurrent recorded as a function of the concentration of peracetic acid inacetate buffer solution 0.1 M pH=5.4, applying, during the amperometrictests a potential of −0.1 V, achieving a sensitivity of 4.21±0.09 nA/μMand RSD %=2%.

From the data obtained, an excellent intra and inter repeatability wasobserved (FIG. 12); the sensor, according to the invention, was able todetect a concentration range of peracetic acid between 20 and 1000 μM(from 1.5 to 76 ppm).

The limit of detection (LOD) and LOQ of the sensor, according to theinvention, were calculated and they resulted to be 1 and 3 μM,respectively.

Finally, the sensor according to the invention also proved itssuitability in pool water. Because pool water is a complex matrix, itwas necessary to dilute the sample and the dilution factor chosen, as acompromise between sensitivity and low matrix effect, was equal to 1:4v/v in buffer solution.

The sensitivity obtained was 6.06±0.03 nA/μM up to 1000 μM.

The accuracy of the sensor was evaluated with the recovery method,obtaining a percentage recovery of 96.4±0.6%, demonstrating the accuracyof the sensor, according to the invention tested.

EXAMPLE 8 Procedure for Preparing a Functionalized Sensor Useful forMeasuring Peracetic Acid Where the Working Electrode and the ReferenceElectrode are Prepared, During the Process of Printing, Using an InkContaining Metal

For the preparation of the functionalized sensor for measuring peraceticacid, the non-functionalized sensor or electrode was used as “startingproduct”, for whose working electrode an ink based on goldmicroparticles was used.

For the measurement, a working solution consisting of 0.05 M acetatebuffer or Britton-Robinson buffer at pH 5.4 and an ionic strength of0.05 M was used; applying to the electrodes a potential of −0.2 V vsAg/AgCl.

Using this electrode linear range and and inter-electrode repeatabilitywere calculated, which were respectively 5-2000 μM (0.4-150 ppm), and10%.

Furthermore, the LOD and LOQ were calculated and they resulted to be 0.8and 2.5 μM, respectively.

EXAMPLE 9 Evaluation of the Interference of Ions Present in the Liquidsto be Analyzed Using the Electrode or Sensor of Example 1

The expert of the art knows that in waters intended for humanconsumption are present ions such as: NO₃ ⁻, SO₄ ²⁻, CO₃ ²⁻, HCO₃ ⁻ andCl⁻, which could interfere in the sensor response; these ions may bepresent in swimming pool waters during maintenance treatments.

The study was performed to assess whether the presence or the absence ofions above mentioned could interfere with the measurement, using thesensor of Example 1.

The results reported in FIG. 7, show that the presence of these ions didnot modify the sensor response to the analyte and especially, the sensordid not show an electrochemical response to them at the concentrationstested

EXAMPLE 10 Printed Electrochemical Sensors and Probes for the ContinuousAnalysis of Chemical Species in In-Line Fluids (Pool Water)

The probes obtained shown in FIGS. 1b-e were connected to the electronicpart to carry out in line measurements. For greater precision andrepeatability of the reading, the probe was inserted into a suitableprobe holder equipped with a flow meter, through which it was possibleto check the liquid flow parameters, such as pressure, flow,temperature, etc., see FIGS. 1b, 1c and 1 d.

1-13. (canceled)
 14. An electrochemical sensor, for the measure influids of an analyte selected from the group consisting of: chlorinedioxide, free chlorine, total chlorine and peracetic acid; saidelectrochemical sensor comprising at least a printed electrodes group;wherein said printed electrodes group is nano and/or micro-structuredusing nano- and/or microparticles selected from the group comprisingcarbon black and/or a metal selected from the group consisting of gold,silver, platinum, copper and combinations or alloys thereof; and whereinsaid printed electrodes group comprises at least a working electrode, atleast a reference electrode, and at least an auxiliary electrode;characterized in that said “printed electrodes group” comprises at leastone hole (21) that allow the gel contained in the reservoir (12) to passthrough.
 15. The electrochemical sensor of claim 14, in which differentelectrode is located in different side of the printed electrode.
 16. Anelectrochemical probe comprising one or more electrochemical sensors ofclaim
 14. 17. Method for preparing the “printed electrodes group” ofclaim 14, wherein said printed electrode group comprises at least aworking electrode, at least a reference electrode, and at least anauxiliary electrode; wherein: the working electrode is prepared andactivated during the process of printing, using an ink containing nano-or microparticles of carbon black and/or nano- or microparticles of ametal selected from the group consisting of gold, silver, platinum,copper and combinations or alloys thereof having an average diameter offrom 20 to 0.05 μm; the auxiliary electrode is prepared and activated,during the process of printing, using an ink containing a carbon basedmaterial; and the reference electrode is prepared and activated, duringthe process of printing, using an ink containing metal microparticlesselected from the group consisting of gold, silver, platinum, copper andcombinations or alloys thereof, having an average diameter of from 20 to0.05 μm.
 18. The method of claim 17, in which the working electrode ofthe “printed electrodes group”, at the end of the printing process isfunctionalised by “drop-casting” by using nanomaterials selected fromthe group consisting of carbon black and/or metallic nanomaterialsselected from the group consisting of gold, silver, platinum, copper andcombinations or alloys thereof.
 19. The method of claim 18, in which thedeposition is made in consecutive applications of 2 μl each.
 20. Themethod of claim 19, in which the metal is gold in microparticles havingan average diameter of 1 μm.
 21. A kit comprising at least anelectrochemical probe of claim 16, and further comprising: at least aprobe holder; at least a capsule; at least an electrolytic solution orgel; at least an hydrophobic and/or hydrophilic membrane; and at leastan electronic control, implementation and/or data transfer.
 22. The kitof claim 21, for the measure of one or more analyte of claim 14, inwater for industrial or civil use.
 23. The kit of claim 21, for themeasure of one or more analyte of claim 14, during a syntheticindustrial process.
 24. The kit of claim 21, for the measure incontinuous of one or more analyte selected from the group consisting of:chlorine dioxide, free chlorine, total chlorine and peracetic acid.